Terminal deoxyribonucleotide transferase mutant, preparation method and kit

Abstract

The invention belongs to the technical field of biosynthesis, and relates to a terminal deoxyribonucleotide transferase mutant, a preparation method and a kit. The mutant is obtained by carrying out multipoint mutation on a truncated wild type terminal deoxyribonucleotide transferase amino acid sequence, and mutation sites comprise R336L, K338G, L397M and E456S or E456G. Compared with the prior art, the method disclosed by the invention has the advantages that the time for rationally transforming the wild type BtTdT is shorter, the transformation efficiency is high, the catalytic efficiency of the obtained mutant is obviously improved, and the mutant has the best activity for catalyzing 3 '-ONH2-dNTP reported at present; and the transformation method of the mutant can be applied to transformation of other TdTs so as to be suitable for improving the catalytic efficiency of the TdTs on other 3 '-block substrates.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering and relates to a terminal deoxyribonucleotidyl transferase mutant and a preparation method and a kit. Background Art

[0002] The vast majority of biological research relies on DNA synthesis, including the synthesis of primers, genes, and even entire chromosomes. DNA synthesis also holds significant potential in information storage. With the explosive growth of information, current silicon-based information storage technologies are no longer sufficient to meet this demand due to their high energy consumption and limited storage capacity. However, DNA is stable, with a half-life of approximately 521 years, consumes less energy to store, and has a data storage capacity several orders of magnitude greater than silicon-based materials. Numerous researchers are working to develop a new generation of DNA-based information storage technologies, in which DNA synthesis is a crucial and essential component.

[0003] Currently, DNA synthesis relies primarily on the well-established chemical solid-phase synthesis method, the phosphoramidite method. Developed in 1981, this method, despite continuous optimization, can only synthesize DNA sequences up to 200-300 bases in length. Longer DNA sequences require the use of DNA assembly techniques. Solid-phase synthesis also has several drawbacks, such as the requirement for large quantities of toxic and hazardous chemical reagents and the resulting wastewater, which can be harmful to the environment.

[0004] Enzymatic DNA synthesis avoids the shortcomings of chemical methods and offers a promising alternative. This process primarily utilizes terminal deoxynucleotidyltransferase (TdT), first discovered in the early 1960s. It is a template-independent DNA polymerase that indiscriminately adds dNTPs to the 3' end of ssDNA, thereby extending the DNA chain. Recent studies have successfully used TdT to synthesize DNA sequences of 10 or 50 nucleotides in length, making the synthesis of longer DNA chains possible.

[0005] The most promising approach to DNA synthesis using TdT enzymatic methods currently utilizes 3'-OH-protected, reversibly terminated dNTPs (RTdNTPs) as extension units, synthesizing DNA in a single-base-per-cycle fashion. However, wild-type TdT, whose natural substrate is dNTP, exhibits low catalytic activity and incomplete conversion of non-natural RTdNTPs, severely limiting the development and industrial application of TdT DNA synthesis processes. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention rationally transforms the wild-type BtTdT and obtains a mutant of BtTdT through point mutation. The transformed BtTdT mutant has greatly improved the catalytic efficiency of the non-natural substrate RTdNTP compared with the wild-type enzyme.

[0007] One of the terminal deoxyribonucleotidyl transferase mutants provided by the present invention is obtained by performing R336L, K338G and L397M point mutations on the truncated wild-type terminal deoxyribonucleotidyl transferase amino acid sequence, and its amino acid sequence is shown in SEQ ID NO.1 in the sequence listing.

[0008] Another terminal deoxyribonucleotidyl transferase mutant provided by the present invention is obtained by performing E456S or E456G point mutation on the amino acid sequence shown in SEQ ID NO. 1 in the sequence listing, and its amino acid sequence is shown in SEQ ID NO. 2 or 3 in the sequence listing.

[0009] The present invention further provides a method for preparing the terminal deoxyribonucleotidyl transferase mutant, comprising the following steps: (1) Truncating the amino acid sequence of the wild-type terminal deoxyribonucleotidyl transferase to remove the BRCT domain; (2) Performing three-point mutations on the truncated amino acid sequence to obtain a mutant with a sequence as shown in SEQ ID NO. 1; or performing four-point mutations to obtain a mutant with an amino acid sequence as shown in SEQ ID NO. 2 or 3.

[0010] Preferably, the three point mutations include R336L, K338G, and L397M.

[0011] Preferably, the four-site mutations include R336L, K338G, L397M and E456S; or R336L, K338G, L397M and E456G.

[0012] Preferably, the mutant containing E456S is obtained by performing structure prediction using a protein structure prediction tool.

[0013] The present invention also provides a nucleic acid encoding the terminal deoxyribonucleotidyl transferase mutant.

[0014] The present invention also provides a kit comprising the nucleic acid or the terminal deoxyribonucleotidyl transferase mutant.

[0015] The beneficial effects of the present invention are as follows: compared with the prior art, the rational transformation of BtTdT by the present invention takes less time and has higher transformation efficiency. The catalytic efficiency of the obtained mutant is significantly improved, and it has the best catalytic activity of 3'-ONH2-dNTP reported so far; and the transformation method of the mutant can be applied to the transformation of other TdTs to improve the catalytic efficiency of TdT for other 3'-block substrates. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The structure of wild-type BtTdT and its activity assay after truncation; a. The two main structural domains of wild-type BtTdT; b. The expression of his-tagged N-terminally truncated BtTdT (BRCT domain removed); c. Activity assay of N-terminally truncated BtTdT, with the ordinate representing relative activity and the abscissa representing the different numbers of amino acids extending the N-terminus of the truncated POLXc domain. Figure 2 Structure prediction and amino acid residue comparison of BtTdT and ZaTdT, as well as activity assays of a series of BtTdT mutants. Figures include: a. Position 397 of BtTdT (green) or BtL397M (magenta); L397 hinders base stacking between the second and third nucleotides at the 3' end of the primer, while the L397M mutation alleviates this hindrance; b. Expression of BtL397M; c. Comparison of key amino acids R336L / K338G in Bt15AA-R336L-K338G (denoted as M2, green) and ZaTdT-R335L-K337G (cyan); d. Expression of BtTdT mutants; e. Extension efficiency of Bt15AA-R336L-K338G-L397M (denoted as M3) with different primers; f. Activity test of BtL397M; g. Activity comparison of BtTdT mutants; Figure 3 Comparison of the different amino acids within 5 Å of the active center between mutants M3 and ZaTdT-R336L-K338G, and the activities after mutation. A. Different amino acids within 5 Å of the active center between M3 (green) and ZaTdT-R336L-K338G (cyan); b. Single and combined mutations of T396A / G or E456A / G and activity testing based on the M3 mutant; c. Extension of M3-E456G to 16 primers and 3'-ONH2-dATP; d. Comparison of the extension efficiency of wild-type BtTdT and mutants to 3'-block-dGTP containing different lengths of blocking groups. Figure 4Comparison of structure and activity after amino acid mutation at position 456; a. When the amino acid at position E456 is mutated based on M3, the distance between the 3'-OH group of the substrate ATP and the enzyme surface changes, E456S (yellow), E456G (cyan), E456 (green), and E456A (magenta); b. Activity determination of the M4 mutant after adding 3'-ONH2-dATP under different time conditions; c. 3'-ONH2-dNTP is added by M4 within 5 minutes. DETAILED DESCRIPTION

[0017] The present invention is described more completely and clearly below with reference to the accompanying drawings and specific embodiments. The embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0018] The present invention provides mutants obtained by modifying the wild-type BtTdT enzyme (amino acid sequence shown in SEQ ID NO. 4 in the sequence listing), wherein the amino acid sequences of the R336L, K338G, and L397M three-point mutants are shown in SEQ ID NO. 1 in the sequence listing; on this basis, the amino acid sequences of the mutants adding E456S or E456G are shown in SEQ ID NO. 2 or 3 in the sequence listing, respectively.

[0019] The process of transforming the wild-type BtTdT enzyme to obtain a mutant is as follows: 1. Truncation of wild-type TdT enzyme The complete structure of wild-type TdT enzyme can be divided into two domains, such as Figure 1 As shown in a, they are the BRCT domain and the POLXc domain, respectively, wherein the BRCT domain is not related to the catalytic activity of TdT, while the core domain containing the POLXc domain has TdT activity. The present invention constructed TdT enzymes of different truncated lengths (removing the BRCT domain) and explored the expression and catalytic activity of different truncated mutants. The mutants of different lengths were expressed and purified, and their catalytic activity towards the substrate dNTP was analyzed. It was found that the catalytic activity of the full-length TdT enzyme was lower than that of the truncated mutants. Moreover, after truncation, the activity was highest when the N-terminus of POLXc was extended to the original 15 amino acid length, while when the number of amino acids extended to the N-terminus was less than 5, the soluble expression level was extremely low when it was expressed in Escherichia coli as a His6-tagged fusion protein, and it basically existed in the form of inclusion bodies, as shown in FIG. Figure 1 As shown in b and c.

[0020] 2. Amino acid residue mutation of BtTdT Before protein engineering, a reliable three-dimensional structure of TdT is required. Therefore, the present invention uses the commonly used prediction method AlphaFold to generate its accurate three-dimensional structure. Since the L398M mutation increases the distance between it and the 3' terminal nucleotide base of the primer, it will reduce the hindrance of the amino acid at this site to the stacking interaction of the 3' terminal nucleotide base of the primer to a certain extent, thereby helping to increase the enzyme reaction rate. Figure 2 As shown in a. The experimental results show that the catalytic efficiency of the L397M mutant for 3'-ONH2-dNTP is about 2 times higher than that of the wild-type BtTdT. Figure 2 As shown in b and f.

[0021] Furthermore, reports have shown that the combined mutations R336L / K338G can improve the enzyme's catalytic activity for the substrate 3'-ONH2-dNTPs. Therefore, the present invention also compared the structures of Bt15AA-R336L-K338G (denoted as M2) and ZaTdT-R335L-K337G. Since the spatial positions of the corresponding residues R336L and K338G after mutation in both residues almost completely overlap, mutations of these two residues in BtTdT are likely to help improve its conversion rate for 3'-ONH2-dNTPs, as shown in Figure 2. Figure 2 The results show that the activity of the double mutant M2 is 5 times higher than that of the wild-type BtTdT, which well verifies the hypothesis of the present invention. The catalytic activity of the triple-site combination mutant of R336L, K338G and L397M for 3'-ONH2-dTTP is further increased by about 60%, as shown in Figure 3c. Figure 2 Subsequently, the present invention tested the catalytic efficiency of the triple point mutant Bt15AA-R336L-K338G-L397M (denoted as M3) for 16 primers (4×4) terminated with different dinucleotides and four 3'-ONH2-dNTP substrates. The catalytic activity was greatly improved. Except for the primers terminated with TC or CC, which had a low conversion rate when adding 3'-ONH2-dATP, all other substrate combinations were basically completely converted within 10 minutes, as shown in Figure 5. Figure 2 As shown in Figure e, this triple point mutant M3 cannot completely convert primers terminated with TC or CC within 10 minutes, so its activity is not high enough and needs further modification to be suitable for efficient enzymatic DNA synthesis.

[0022] Since ZaTdT-R336L-K338G has a complete extension for the addition of 3'-ONH2-dATP to primers ending with TC, in order to further improve the catalytic ability of the BtTdT mutant for the addition of 3'-ONH2-dATP, the present invention compared the key amino acid residues within 5 Å of the catalytic center of ZaTdT-R336L-K338G and mutant M3, such as Figure 3 As shown in a. Obviously, T396 and E456 in mutant M3 are the different amino acids between the two, and are replaced by A and G respectively in ZaTdT-R336L-K338G. The present invention speculates that the substitution of these two smaller residues may provide a larger activity space for 3'-ONH2-dATP. Therefore, T396G / A and E456A / G mutations were constructed based on M3 to detect their effects on the catalytic efficiency of 3'-ONH2-dATP. All four mutants showed higher conversion rates than the parent mutant M3, especially the E456G mutant showed the best catalytic effect, which could completely convert primers terminated with TC and CC within 10 and 20 min, as shown in Figure 2. Figure 3 As shown in b. Subsequently, a combination of mutations was performed on E456G and T396G / A. However, compared with the four-point mutation M3-E456G, the five-point mutant did not further enhance the catalytic activity, as shown in Figure 3 As shown in b. At the same time, the addition efficiency of M3-E456G to 16 primer pairs of 3'-ONH2-dATP was determined, confirming that the increase in its activity would not reduce its catalytic ability to other substrates, such as Figure 3 As shown in Figure c. The results showed that only primers ending in CC could not be fully converted within 10 minutes. This "shortest board" needs to be further improved through enzyme engineering to obtain better mutants.

[0023] 3. Rational design based on the protein prediction tool AlphaFold 3 (AF3) Considering that site E456 is crucial to the catalytic efficiency of 3'-block dNTP, increasing the pocket size to provide greater freedom for the nucleotide can improve the catalytic efficiency. In addition, the present invention believes that there may be more suitable amino acid substitutions for the E456 site, which will increase the distance between the 3'-ONH2 group and the enzyme surface, thereby giving the substrate better freedom and forming non-covalent bonds such as hydrogen bonds with surrounding amino acids that are more conducive to the reaction, thereby obtaining higher catalytic activity. At the same time, in order to reduce the workload and shorten the research time, the present invention uses the most advanced protein structure prediction tool AF3 to predict the structure of mutants with 20 amino acid mutations at site 456 in M3. Since AF3 can provide ligands such as metal ions or DNA chains during docking, ATP, an alternative substrate to 3'-ONH2-dATP, is selected as the ligand during prediction. The modeling results show that only in the E456S mutant is the 3'-OH group of the substrate mononucleotide the farthest from the enzyme surface, which indicates that the E456S mutation may further increase the catalytic activity of the enzyme, such as Figure 4As shown in Figure a. The M3-E456S (denoted as M4) mutant was expressed and purified, and then its extension efficiency for 3'-ONH2-dNTP was tested. The results showed that the mutant M4 was able to achieve complete substrate conversion and shorten the reaction time to 5 minutes. Figure 4 When tested with 3'-ONH2-dATP for a 1-min extension time, all three primers (terminated with TC, CT, or CC) failed to fully convert. Furthermore, the catalytic activity of M4 towards three other 3'-ONH2-dNTPs was also tested, and the results showed that all 64 substrate pairs were fully converted within 5 minutes. Figure 4 As shown in c.

Claims

1. A terminal deoxyribonucleotidyl transferase mutant, characterized in that: The mutant is obtained by performing point mutations R336L, K338G and L397M on the truncated wild-type terminal deoxyribonucleotidyl transferase amino acid sequence, and its amino acid sequence is shown in SEQ ID NO.1 in the sequence table.

2. The terminal deoxyribonucleotidyl transferase mutant according to claim 1, characterized in that: The mutant is obtained by performing E456S or E456G point mutation on the amino acid sequence shown in SEQ ID NO. 1 in the sequence listing, and its amino acid sequence is shown in SEQ ID NO. 2 or 3 in the sequence listing, respectively.

3. The method for preparing a terminal deoxyribonucleotidyl transferase mutant according to claim 1 or 2, wherein: The following steps are involved: (1) Truncating the amino acid sequence of the wild-type terminal deoxyribonucleotidyl transferase to remove the BRCT domain; (2) Performing three-point mutations on the truncated amino acid sequence to obtain a mutant with an amino acid sequence as shown in SEQ ID NO. 1; or performing four-point mutations to obtain a mutant with an amino acid sequence as shown in SEQ ID NO. 2 or 3.

4. The method for preparing a terminal deoxyribonucleotidyl transferase mutant according to claim 3, wherein: The three point mutations include R336L, K338G, and L397M.

5. The method for preparing a terminal deoxyribonucleotidyl transferase mutant according to claim 3, wherein: The four-site mutations include R336L, K338G, L397M and E456S; or R336L, K338G, L397M and E456G.

6. The method for preparing a terminal deoxyribonucleotidyl transferase mutant according to claim 5, characterized in that: The mutant containing E456S was obtained by structural prediction using a protein structure prediction tool.

7. A nucleic acid, characterized in that The nucleic acid encodes the terminal deoxyribonucleotidyl transferase mutant according to claim 1 or 2.

8. A kit, characterized in that Comprising the nucleic acid of claim 7.

9. A kit, characterized in that The invention comprises the terminal deoxyribonucleotidyl transferase mutant according to claim 1 or 2.

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